PROJECT SUMMARY Central Nervous System disorders have proven to be among the most difficult of diseases to treat and have placed an enormous burden on society. A major contributing factor to this situation is that currently we often cannot distinguish between normal and pathological states since we do not possess even a basic 'ground truth' map of how neurons and other cells are arranged at the nanoscale and macroscale level for representative pieces of neuronal tissue or whole organ. Extensive efforts are underway to build these fine scale structural maps of mammalian brains with the hope that these will lead to an understanding of the how brain ultimately functions and how best to target therapeutics. However the problem is immense as a single cubic mm of brain tissue contains about 100,000 neurons and roughly a billion synaptic connections and some of these neurons are thought to span across the brain. The overwhelming majority of these structural details are too small to be resolved with even the best optical microscopes and current super resolution approaches lack the necessary throughput. We propose to meet this challenge by developing an ultra-high speed Serial Two-Photon Tomography system that combines high speed resonant scanning with TissueVision's novel multi-focal microscopy, and which will be able to quickly image 3D volumes of neuronal tissue with sub-micron resolution. We will further combine this with TissueVision?s novel automated slice collector module that automatically captures and mounts tissue sections onto slides. Working with our collaborators at the Allen Institute for Brian Science and at MIT we will apply this system to study the CNS cytoarchitecture by producing whole brain axonal reconstructions through the nano to macroscale. We will employ a recent technique the Boyden lab has developed called Expansion Microscopy (ExM) that physically expands tissue up to 8-fold and provides an effective nanoscale (~25nm) view into the structure of macroscopic portions of the CNS, and use this technique to aid axonal reconstructions of whole mouse brains from a novel suite of transgenic mouse lines that the Allen Institute has produced that target specific cell types. For this project, we have put together team of leading experts who have deep expertise with optics and neuroscience as well as a successful track record of commercializing biomedical imaging systems for the neuroscience market. While our focus here is on nanoscale imaging of CNS tissues, the device we will develop will represent a major tool for a variety of brain mapping efforts including whole brain neuronal circuit analysis and mesoscale mapping efforts.